Alkaline phosphatase (orthrophosphoric-monoester phosphohydrolase, alkaline optimum, EC. 3.1.3.1) is an enzyme widely distributed in nature. The enzyme has been isolated from a variety of eukaryotic sources such as human placenta, liver, bone, leukocytes and serum; bovine bone, intestine (particularly calf intestinal) and kidney; and rat liver; as well as a variety of prokaryotic sources including Escherichia coli, Bacillus subtilis, Bacillus, lichenoformis, Micrococcus sodonensis, Thermoactinomyces, vulgaris, and Lysobacter enzymogenes.
The enzymes have been reasonably well characterized and all appear to be Zn(II) metalloenzymes which catalyze the hydrolysis of monoesters by means of the formation of a phosphoseryl intermediate. A number of excellent literature reviews exist (See for example: Reid, T. W. and I. B. Wilson, Enzymes 4: 737 (1971); Coleman, J. E. and J. F. Chlebowski, Adv. Inorg. Biochem. 1:1 (1979); McComb, R. B. et al. "Alkaline Phosphatase", Plenum Press, New York, (1979); Coleman, J. E. and P. Gettins, Adv. in Enzymol. 55: 381 (1983); and Wyckoff, H. W. et al., Adv. in Enzmol. 55: 453 (1983)).
The enzyme from E. coli has been particularly well studied and complete amino acid sequence as well as three dimensional structural data are available. The enzyme exists as a dimer of approximately 94,000 molecular weight, composed of two identical approximately 47,000 molecular weight monomer subunits. The monomer is an unglycosylated single chain of 449 amino acids in its mature form. The enzyme is localized in the periplasmic space and as is common with such extra-membrane protein products, it is initially synthesized on membrane bound polysomes in a precursor form and secreted through the membrane with the assistance of a signal peptide region which is cleared from the enzyme as a consequence of its deposition within the periplasmic region.
In its native form, the protein possesses remarkable stability as reflected by its resistance to thermal and chemical denaturation (Chlebowski, J. F., et al., J. Biol. Chem. 252: 7053 (1977)). The enzyme has also previously been reported to be resistant to proteolytic modification (Schlesinger, M. J. et al. Ann. N.Y. Acad. Sci. 166: 368 (1969); Reid I M and I. B. Wilson, The Enzymes 4: 373 (1971). Stabilization of the protein is due principally to the association of Zn(II) and Mg(II) ions in the holoenzyme. As isolated from the periplasmic space of the E. coli bacterium, the enzyme has bound up to 4 eq of Zn(II) and 2 eq of Mg(II). Three metal ion-binding sites, designated A, B, and C, are located on each subunit in a cluster, lying within 4 to 7 A.degree. of one another. Since the binding of a minimum of 2 eq of Zn(II)/dimer is required for activity (phosphate monoester hydrolysis), the metal ion cluster appears to define the active site region. The location of Ser-102, which is covalently phosphorylated in the course of the reaction, at the metal ion cluster permits an unequivocal location of the active center.
The protein is reported to display cooperative subunit interactions affecting metal ion association with the metal-free apoenzyme and ligand association with the active metalloenzyme. Since the active centers of the holoenzyme lie 32 .ANG..degree. apart across the 2-fold symmetry axis relating the subunits of the dimer, such cooperative effects would appear to involve the transmission of conformational information through the interconnecting polypeptide structure. Consistent with this depiction is the extensive array of intersubunit contacts at the monomer-monomer interface. The existence of cooperative phenomena has, however, been a source of continuous controversy in the literature. This has, at least in part, detracted from the plausibility of allosteric interactions as playing a role in modulating the structure and reactivity of the enzyme.
As mentioned above, the effects of certain ions on the stability of the enzyme have been studied in some detail not only with respect to the E. coli enzyme but also with respect to a number of different alkaline phosphatases isolated from a variety of sources.
Ensinger, et al. (Biochem. et Biophys. Acta. 527: 432 (1978)) discloses the inactivation of calf intestine alkaline phosphatase by chelating agents. The inactivation was shown to be reversible (i.e., the activity was restored by readdition of Zn.sup.++) at pH 8.0. It was also shown that, at more alkaline pHs, the inactivation became irreversible and that complete irreversible inactivation occurred at pH 9.8.
In an investigation of structural-functional domains of bacterial alkaline phosphatase, McCracken and Meighen (J. Biol. Chem. 256 (8): 3945 (1981)) provide evidence that certain histidine residues are responsible for meta ion binding and that by chemically blocking (derivatizing) the histidine moieties, the stability of the enzyme subunit structure is affected.
Sinha et al. (Indian J. Exp. Biol. 19: 453 (1981)) disclose the cation requirements of an alkaline phosphatase from a thermophile, Thermoactinomyces vulgaris. These researchers demonstrate that the presence of Mg.sup.++ is necessary.
Ueda (Biol. J. Clin. Pathol. 80(3): 342 (1983)) discloses certain physiocochemical properties of an alkaline phosphatase isolated from leukocytes. The enzyme is relatively heat labile, being 100% inactivated at 56.degree. C. after 2 minutes whereas the placental form of alkaline phosphatase is 100% stable at the same temperature after 15 minutes. The leukocyte alkaline phosphatase is inhibited by 50% after incubation with 0.02M EDTA.
Yamashita, et al. (J. Biochem. (JAPAN) 80: 129 (1976) teach that tryptic digestion of serum protects human serum alkaline phosphatase from histidine-mediated heat inactivation. The authors show that trypsin does not affect the heat stability of alkaline phosphatase in the absence of histidine.
In a recent report vonTigerstrom (Appl. and Environ. Microbiol. 47 (4): 693 (1984) discloses a potential new source of alkaline phosphatase. One of the forms of the enzyme is apparently extracellular. The Lysobacter enzyme can be distinguished from other bacterial alkaline phosphatases and calf intestine alkaline phosphatases in that chelating agents have little or no effect.
In direct contrast to the reports of Schlesinger et al. (supra) and Yamashita et al. (supra), it has now been surprisingly discovered that the alkaline phosphatase from E. coli is susceptibile to modification by trypsin. It is further demonstrated that the modified enzyme displays unique properties that render the modified enzyme particularly useful in a variety of enzymological procedures.